Persistent pulmonary hypertension of the newborn (PPHN) is a serious clinical problem that occurs when pulmonary vascular resistance does not decrease normally during the transition to gas breathing at birth. We have reported that activity of endothelial nitric oxide synthase (NOS) and sGC are decreased in a lamb model of PPHN, but the specific mechanisms producing these changes have not been well characterized. Although inhaled nitric oxide is now standard therapy infants with PPHN, the beneficial effects are minimal or transient in almost 50% of treated infants. Therefore, a better understanding of the mechanisms involved in the development of PPHN and new treatment strategies are urgently needed. We have preliminary data that strongly suggest: 1) a role for reactive oxygen species (ROS) in the pathogenesis of PPHN; and 2) ROS scavengers, such as recombinant human superoxide dismutase (rhSOD) dramatically improve oxygenation. Therefore, this grant proposal will focus on the role of ROS in PPHN, and whether treatment with specific scavengers such as rhSOD might reverse the vascular changes of PPHN. A lamb model of PPHN will be studied created by antenatal ligation of the ductus arteriosus. This model has many of the anatomic and physiologic alterations observed in human infants with PPHN. We propose two specific aims: (1) To examine the role of ROS in promoting pulmonary vascular reactivity in PPHN, and (2) To determine the mechanisms of SOD-mediated amelioration of PPHN. To accomplish the first specific aim, we will use a combination of in vivo and in vitro approaches to better characterize the specific ROS that are produced in PPHN lambs following delivery and mechanical ventilation, and test the hypothesis that superoxide and hydrogen peroxide derived from NADPH oxidase promote pulmonary vascular reactivity in PPHN lambs. To accomplish specific aim 2, we will primarily use in vivo studies to test the hypothesis that scavenging superoxide with rhSOD increases endogenous NO production via changes in NOS expression and/or activity. [unreadable] [unreadable]